黃花蜜菜藥酒萃取物對人類多型性神經膠母細胞瘤細胞株之影響與相關訊息機制研究

The effect and mechanism of Wedelia chinensis extract on human glioblastoma multiforme cells

葉沛蓉

黃花蜜菜 ； 神經膠母細胞瘤 ； 腦癌 ； Wedelia chinensis ； glioblastoma ； brain cancer

中山醫學大學生化微生物免疫研究所學位論文

2017年

碩士

曾博修

繁體中文

多型性神經膠母細胞瘤 (glioblastoma multiforme，GBM) 是最常見且最具侵襲性的腦瘤，又稱為膠質細胞瘤 (glioblastoma)、第四級星形細胞瘤 (grade IV astrocytoma)，治療方式為手術切除後再與放射治療和化學治療同時治療。膠質細胞瘤產生的位置難以用手術完全去除，並且放射治療無可避免對重要腦區造成任何損傷。因此，對於惡性膠質瘤的治療方式是重要的課題。黃花蜜菜 (Wedelia chinensis (Osbeck) Merr.) 是台灣傳統的菊科中草藥，已經被證實具有抗發炎性質、保護肝臟的效果，並具有神經藥理學活性。過去文獻指出，黃花蜜菜萃取物可以抑制前列腺癌及乳癌細胞生長。但目前對於黃花蜜菜萃取物抑制膠質瘤細胞生長的機制尚未明確，因此本篇研究利用黃花蜜菜藥酒萃取物進行人類神經膠母細胞瘤細胞株之影響與相關訊息機制研究。 　　本篇研究以細胞存活率試驗證實黃花蜜菜萃取物確實會抑制人類神經膠母細胞瘤細胞株 (GBM8401和U-87MG) 生長，並且抑制GBM8401細胞移動能力。此外在GBM8401細胞處理黃花蜜菜萃取物發現Sub G1有增加的情形，同時也觀察到p21蛋白表現增加、cyclin D1蛋白表現減少；與內源性凋亡路徑的相關蛋白表現中，抗凋亡蛋白Bcl-2 蛋白表現減少，促凋亡蛋白Bax、Cytochrome C、Apaf-1、Caspase-9及Caspase-3蛋白表現增加。由這些實驗結果顯示黃花蜜菜萃取物可誘導GBM8401細胞細胞週期停滯和凋亡，並表明黃花蜜菜在未來可能有助於提供GBM治療藥物的替代選擇。

Glioblastoma multiforme (GBM), known as glioblastoma and grade IV astrocytoma, is the most common and most aggressive brain cancer with the worst prognosis and survival time. Standard treatment for GBM includes surgical resection, combination with radiotherapy and chemotherapy. However, the anatomic location of gliomas makes it very difficult to surgically remove the tumor and it is impossible to avoid any damage to vital brain regions with radiotherapy. Therefore, therapeutic strategies for malignant glioma are urgently required improved. Wedelia chinensis is known as a traditional Chinese herbal medicine has been shown to have anti-inflammatory properties, anti-hepatic toxicity, and neuropharmacological activity. Recent evidences have suggested that Wedelia chinensis extract inhibits the growth of prostate cancer and breast cancer cells. However, little is known about the effects of Wedelia chinensis extract on glioblastoma. We herein intend to investigate the effect of Wedelia chinensis extract on human glioblastoma cells. 　　In this study, significantly decreased survival ratios of GBM8401 and U-87MG cells were detected in the presence of Wedelia chinensis extract. Significantly decreased migration was detected in GBM8401 cells treated with Wedelia chinensis extract. Additionally, significantly increased sub-G1 population was observed in GBM8401 cells that were treated with Wedelia chinensis extract as well the significantly increased P21 and decreased cyclin D1 proteins, significantly decreased Bcl-2 (anti-apoptotic) proteins and increased Bax (pro-apoptotic), Cytochrome C, Apaf-1, Caspase-9, Caspase-3 were detected by Western blot analysis. Altogether, these results demonstrated that treatment of GBM8401 cells with Wedelia chinensis extract induced cell cycle arrest and apoptosis, suggesting Wedelia chinensis as a useful alternative choice in treatment of GBM.

1. 1. 104年行政院衛生署癌症統計
   連結：
2. 3. Furnari FB, Fenton T, Bachoo RM, Mukasa A, Stommel JM, Stegh A, Hahn WC, Ligon KL, Louis DN, Brennan C, Chin L, DePinho RA and Cavenee WK (2007) Malignant astrocytic glioma: genetics, biology, and paths to treatment. Genes Dev 21:2683-710. doi: 10.1101/gad.1596707
   連結：
3. 4. van Tellingen O, Yetkin-Arik B, de Gooijer MC, Wesseling P, Wurdinger T and de Vries HE (2015) Overcoming the blood-brain tumor barrier for effective glioblastoma treatment. Drug Resist Updat 19:1-12. doi: 10.1016/j.drup.2015.02.002
   連結：
4. 5. Faraz S, Pannullo S, Rosenblum M, Smith A and Wernicke AG (2016) Long-term survival in a patient with glioblastoma on antipsychotic therapy for schizophrenia: a case report and literature review. Ther Adv Med Oncol 8:421-428. doi: 10.1177/1758834016659791
   連結：
5. 6. Manrow RE, Beckwith M and Johnson LE (2014) NCI's Physician Data Query (PDQ(R)) cancer information summaries: history, editorial processes, influence, and reach. J Cancer Educ 29:198-205. doi: 10.1007/s13187-013-0536-3
   連結：
6. 7. Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee WK, Ohgaki H, Wiestler OD, Kleihues P and Ellison DW (2016) The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary. Acta Neuropathol 131:803-20. doi: 10.1007/s00401-016-1545-1
   連結：
7. 8. Ostrom QT, Gittleman H, Fulop J, Liu M, Blanda R, Kromer C, Wolinsky Y, Kruchko C and Barnholtz-Sloan JS (2015) CBTRUS Statistical Report: Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2008-2012. Neuro Oncol 17 Suppl 4:iv1-iv62. doi: 10.1093/neuonc/nov189
   連結：
8. 9. Preusser M, de Ribaupierre S, Wohrer A, Erridge SC, Hegi M, Weller M and Stupp R (2011) Current concepts and management of glioblastoma. Ann Neurol 70:9-21. doi: 10.1002/ana.22425
   連結：
9. 10. Dunn GP, Rinne ML, Wykosky J, Genovese G, Quayle SN, Dunn IF, Agarwalla PK, Chheda MG, Campos B, Wang A, Brennan C, Ligon KL, Furnari F, Cavenee WK, Depinho RA, Chin L and Hahn WC (2012) Emerging insights into the molecular and cellular basis of glioblastoma. Genes Dev 26:756-84. doi: 10.1101/gad.187922.112
   連結：
10. 11. Kaufmann JK and Chiocca EA (2014) Glioma virus therapies between bench and bedside. Neuro Oncol 16:334-51. doi: 10.1093/neuonc/not310
    連結：
11. 12. Huang YC, Wei KC, Chang CH, Yang JT, Ho JT, Shen CC, Su CF, Cho DY, Ma HI, Lin JW, Lee EJ, Wu JJ, Tsai MD, Chang CK, Howng SL and Chang CN (2011) A retrospective survey of patients with malignant gliomas treated in the neuro-oncological care system under the Universal National Health Insurance program in Taiwan. J Clin Neurosci 18:784-8. doi: 10.1016/j.jocn.2010.10.016
    連結：
12. 13. Norden AD and Wen PY (2006) Glioma therapy in adults. Neurologist 12:279-92. doi: 10.1097/01.nrl.0000250928.26044.47
    連結：
13. 14. Park DM, Sathornsumetee S and Rich JN (2010) Medical oncology: treatment and management of malignant gliomas. Nat Rev Clin Oncol 7:75-7. doi: 10.1038/nrclinonc.2009.221
    連結：
14. 15. Blakeley J (2008) Drug delivery to brain tumors. Curr Neurol Neurosci Rep 8:235-41.
    連結：
15. 16. Lin SC, Lin CC, Lin YH and Shyuu SJ (1994) Hepatoprotective effects of Taiwan folk medicine: wedelia chinensis on three hepatotoxin-induced hepatotoxicity. Am J Chin Med 22:155-68. doi: 10.1142/S0192415X9400019X
    連結：
16. 17. Prakash T, Rao NR and Swamy AH (2008) Neuropharmacological studies on Wedelia calendulacea Less stem extract. Phytomedicine 15:959-70. doi: 10.1016/j.phymed.2008.05.005
    連結：
17. 18. Haldar PK, Bhattacharya S, Dewanjee S and Mazumder UK (2011) Chemopreventive efficacy of Wedelia calendulaceae against 20-methylcholanthrene-induced carcinogenesis in mice. Environ Toxicol Pharmacol 31:10-7. doi: 10.1016/j.etap.2010.08.003
    連結：
18. 19. Huang YT, Wen CC, Chen YH, Huang WC, Huang LT, Lin WC, Arulselvan P, Liao JW, Lin SH, Hsiao PW, Kuo SC and Yang NS (2013) Dietary uptake of Wedelia chinensis extract attenuates dextran sulfate sodium-induced colitis in mice. PLoS One 8:e64152. doi: 10.1371/journal.pone.0064152
    連結：
19. 20. Tsai CH, Lin FM, Yang YC, Lee MT, Cha TL, Wu GJ, Hsieh SC and Hsiao PW (2009) Herbal extract of Wedelia chinensis attenuates androgen receptor activity and orthotopic growth of prostate cancer in nude mice. Clin Cancer Res 15:5435-44. doi: 10.1158/1078-0432.CCR-09-0298
    連結：
20. 21. Tsai CH, Tzeng SF, Hsieh SC, Lin CY, Tsai CJ, Chen YR, Yang YC, Chou YW, Lee MT and Hsiao PW (2015) Development of a standardized and effect-optimized herbal extract of Wedelia chinensis for prostate cancer. Phytomedicine 22:406-14. doi: 10.1016/j.phymed.2015.01.013
    連結：
21. 22. Lin FM, Chen LR, Lin EH, Ke FC, Chen HY, Tsai MJ and Hsiao PW (2007) Compounds from Wedelia chinensis synergistically suppress androgen activity and growth in prostate cancer cells. Carcinogenesis 28:2521-9. doi: 10.1093/carcin/bgm137
    連結：
22. 23. Lee WJ, Chen WK, Wang CJ, Lin WL and Tseng TH (2008) Apigenin inhibits HGF-promoted invasive growth and metastasis involving blocking PI3K/Akt pathway and beta 4 integrin function in MDA-MB-231 breast cancer cells. Toxicol Appl Pharmacol 226:178-91. doi: 10.1016/j.taap.2007.09.013
    連結：
23. 24. Lee YJ, Lin WL, Chen NF, Chuang SK and Tseng TH (2012) Demethylwedelolactone derivatives inhibit invasive growth in vitro and lung metastasis of MDA-MB-231 breast cancer cells in nude mice. Eur J Med Chem 56:361-7. doi: 10.1016/j.ejmech.2012.07.041
    連結：
24. 25. Yang MY, Wang CJ, Chen NF, Ho WH, Lu FJ and Tseng TH (2014) Luteolin enhances paclitaxel-induced apoptosis in human breast cancer MDA-MB-231 cells by blocking STAT3. Chem Biol Interact 213:60-8. doi: 10.1016/j.cbi.2014.02.002
    連結：
25. 26. Kim B, Jung N, Lee S, Sohng JK and Jung HJ (2016) Apigenin Inhibits Cancer Stem Cell-Like Phenotypes in Human Glioblastoma Cells via Suppression of c-Met Signaling. Phytother Res 30:1833-1840. doi: 10.1002/ptr.5689
    連結：
26. 27. Tashima Y, Hamada H, Okamoto M and Hanai T (2008) Prediction of key factor controlling G1/S phase in the mammalian cell cycle using system analysis. J Biosci Bioeng 106:368-74. doi: 10.1263/jbb.106.368
    連結：
27. 28. Smith ML and Fornace AJ, Jr. (1996) Mammalian DNA damage-inducible genes associated with growth arrest and apoptosis. Mutat Res 340:109-24.
    連結：
28. 29. Zhao Hongmei (2012) Extrinsic and Intrinsic Apoptosis Signal Pathway Review, Apoptosis and Medicine, Dr. Tobias Ntuli (Ed.), InTech, doi: 10.5772/50129.
    連結：
29. 30. Ho SY, Huang PC, Guo HR, Chang WH, Chen RJ, Wei BL, Wu WJ, Tai C and Wang YJ (2006) Mechanisms of apoptosis induction and cell cycle regulation in irradiated leukemia U937 cells and enhancement by arsenic trioxide. Radiat Res 165:390-9.
    連結：
30. 31. Wlodkowic D, Telford W, Skommer J and Darzynkiewicz Z (2011) Apoptosis and beyond: cytometry in studies of programmed cell death. Methods Cell Biol 103:55-98. doi: 10.1016/B978-0-12-385493-3.00004-8
    連結：
31. 32. Li J and Yuan J (2008) Caspases in apoptosis and beyond. Oncogene 27:6194-206. doi: 10.1038/onc.2008.297
    連結：
32. 33. Liu T, Ren D, Zhu X, Yin Z, Jin G, Zhao Z, Robinson D, Li X, Wong K, Cui K, Zhao H and Wong ST (2013) Transcriptional signaling pathways inversely regulated in Alzheimer's disease and glioblastoma multiform. Sci Rep 3:3467. doi: 10.1038/srep03467
    連結：
33. 34. Yamamoto T, Fukaya C, Obuchi T, Watanabe M, Ohta T, Kobayashi K, Oshima H and Yoshino A (2016) Glioblastoma Multiforme Developed during Chronic Deep Brain Stimulation for Parkinson Disease. Stereotact Funct Neurosurg 94:320-325. doi: 10.1159/000448925
    連結：
34. 35. Lee WH, Jin JS, Tsai WC, Chen YT, Chang WL, Yao CW, Sheu LF and Chen A (2006) Biological inhibitory effects of the Chinese herb danggui on brain astrocytoma. Pathobiology 73:141-8. doi: 10.1159/000095560
    連結：
35. 36. Lin J, Chen L, Lin Z and Zhao M (2007) Inhibitory effect of triptolide on glioblastoma multiforme in vitro. J Int Med Res 35:490-6.
    連結：
36. 37. Mimura J, Kosaka K, Maruyama A, Satoh T, Harada N, Yoshida H, Satoh K, Yamamoto M and Itoh K (2011) Nrf2 regulates NGF mRNA induction by carnosic acid in T98G glioblastoma cells and normal human astrocytes. J Biochem 150:209-17. doi: 10.1093/jb/mvr065
    連結：
37. 38. Urbanska K, Sokolowska J, Szmidt M and Sysa P (2014) Glioblastoma multiforme - an overview. Contemp Oncol (Pozn) 18:307-12. doi: 10.5114/wo.2014.40559
    連結：
38. 2. BCRC生物資源保存及研究中心線上目錄